This invention relates to multipotent neuroepithelial stem cells, lineage-restricted intermediate precursor cells, and methods of making thereof More particularly, the invention relates to neuroepithelial stem cells that retain the capabilities of self-renewal and differentiation into neurons, astrocytes, and oligodendrocytes. Further, the invention relates to oligodendrocyte-astrocyte-restricted precursor cells that are capable of self-renewal and differentiation into astrocytes and oligodendrocytes, but not neurons. Methods of generating, isolating, and culturing such neuroepithelial stem cells and oligodendrocyte-astrocyte precursor cells are also described. Still further, the invention relates to a methods of generating, isolating, and culturing neural crest stem cells and derivatives of the peripheral nervous system from neuroepithelial stem cells.
Multipotent cells with the characteristics of stem cells have been identified in several regions of the central nervous system and at several developmental stages. F. H. Gage et al., Isolation, Characterization and Use of Stem Cells from the CNS, 18 Ann. Rev. Neurosci. 159-92 (1995); M. Marvin and R. McKay, Multipotential Stem Cells in the Vertebrate CNS, 3 Semin. Cell. Biol. 401-11 (1992); R. P. Skoff, The Lineages of Neuroglial Cells, 2 The Neuroscientist 335-44 (1996). These cells, often referred to as neuroepithelial stem cells (NEP cells), have the capacity to undergo self renewal and to differentiate into neurons, oligodendrocytes, and astrocytes, thus representing multipotent stem cells. A. A. Davis and S. Temple, A Self-Renewing Multipotential Stem Cell in Embryonic Rat Cerebral Cortex, 362 Nature 363-72 (1994); A. G. Gritti et al., Multipotential Stem Cells from the Adult Mouse Brain Proliferate and Self-Renew in Response to Basic Fibroblast Growth Factor, 16 J. Neurosci. 1091-1100 (1996); B. A. Reynolds et al., A Multipotent EGF-Responsive Striatal Embryonic Progenitor Cell Produces Neurons and Astrocytes, 12 J. Neurosci. 4565-74 (1992); B. A. Reynolds and S. Weiss, Clonal and Population Analyses Demonstrate that an EGF-Responsive Mammalian Embryonic CNS Precursor is a Stem Cell, 175 Developmental Biol. 1-13 (1996); B. P. Williams et al., The Generation of Neurons and Oligodendrocytes from a Common Precursor Cell, 7 Neuron 685-93 (1991).
The nervous system also contains precursor cells with restricted differentiation potentials. T. J. Kilpatrick and P. F. Bartlett, Cloned Multipotential Precursors from the Mouse Cerebrum Require FGF-2, Whereas Glial Restricted Precursors are Stimulated with Either FGF-2 or EGF, 15 J. Neurosci. 3653-61 (1995); J. Price et al., Lineage Analysis in the Vertebrate Nervous System by Retrovirus-Mediated Gene Transfer, 84 Developmental Biol. 156-60 (1987); B. A. Reynolds et al., supra; B. A. Reynolds and S. Weiss, supra; B. Williams, Precursor Cell Types in the Germinal Zone of the Cerebral Cortex, 17 BioEssays 391-93 (1995); B. P. Williams et al., supra. The relationship between multipotent stem cells and lineage restricted precursor cells is still unclear. In principal, lineage restricted cells could be derived from multipotent cells, but this is still a hypothetical possibility in the nervous system with no direct experimental evidence.
During development, the neuroepithelial cells that comprise the caudal neural tube differentiate into neurons and glia. Neurons arise from neuroepithelial precursors first and eventually develop unique phenotypes defined by their trophic requirements, morphology, and function. Motoneurons are among the first neurons to develop. V. Hamburger, The Mitotic Patterns in the Spinal Cord of the Chick Embryo and Their Relationship to the Histogenic Process, 88 J. Comp. Neurol. 221-84 (1948); H. O. Nornes and G. D. Das, Temporal Pattern of Neurogenesis in the Spinal Cord of Rat. 1. Time and Sites of Origin and Migration and Settling Patterns of Neuroblasts, 73 Brain Res. 121-38 (1974); J. Altman and S. Bayer, The Development of the Rat Spinal Cord, 85 Adv. Anat. Embryol. Cell Biol. 32-46 (1984); P. E. Phelps et al., Generation Patterns of Four Groups of Cholinergic Neurons in Rat Cervical Spinal Cord: A Combined Tritiated Thymidine Autoradiographic and Choline Acetyltransferase Immunocytochemical Study, 273 J. Comp. Neurol. 459-72 (1988); P. E. Phelps et al., Embryonic Development of Four Subsets of Cholinergic Neurons in Rat Cervical Spinal Cord, 291 J. Comp. Neurol. 9-26 (1990). Motoneurons can be distinguished from other neurons present in the spinal cord by their position and the expression of a number of specific antigens. E. W. Chen and A. Y. Chiu, Early Stages in the Development of Spinal Motor Neurons, 320 J. Comp. Neurol. 291-303 (1992). Tag-1, J. Dodd et al., Spatial Regulation of Axonal Glycoprotein Expression on Subsets of Embryonic Spinal Neurons, 1 Neuron 105-16 (1988), islet-1, J. Erickson et al., Early Stages of Motor Neuron Differentiation Revealed by Expression of Homeobox Gene Islet-1, 256 Science 1555-59 (1992), and p75, W. Camu and C. E. Henderson, Purification of Embryonic Rat Motorneurons by Panning on a Monoclonal Antibody to the Low-Affinity NGF Receptor, 44 J. Neurosci. 59-70 (1992), are expressed uniquely on rat and chick motoneurons early in their development, but are not detectable on other spinal cord cells and, therefore, may serve to distinguish motoneurons from other neural tube cells. Astrocytes, characterized by glial fibrillary acidic protein (GFAP) immunoreactivity, appear soon after; GFAP staining is seen at embryonic day 16 (E16). M. Hirano and J. E. Goldman, Gliogenesis in the Rat Spinal Cord: Evidence for the Origin of Astrocytes and Oligodendrocytes from Radial Precursors, 21 J. Neurosci. Res. 155-67 (1988). Astrocytic cells proliferate and populate the gray and white matter of the spinal cord, and both type 1 and type 2 astrocytes have been identified in the spinal cord. B. C. Warf et al., Evidence for the Ventral Origin of Oligodendrocytic Precursors in the Rat Spinal Cord, 11 J. Neurosci. 2477-88 (1991). Oligodendrocytes appear later and are first detected around birth, though oligodendrocyte precursors may be present as early as E14 based on platelet derived growth factor alpha-receptor (PDGFRA) expression and culture assays. N. P. Pringle and W. D. Richardson, A Singularity of PDGF Alpha-Receptor Expression in the Dorsoventral Axis of the Neural Tube May Define the Origin of the Oligodendrocyte Lineage, 117 Development 525-33 (1993); B. C. Warf et al., supra.
As will be shown herein, NEP cells grow on fibronectin and require fibroblast growth factor (FGF) and an as yet uncharacterized component present in chick embryo extract (CEE) to proliferate and maintain an undifferentiated phenotype in culture. The growth requirements of NEP cells are different from neurospheres isolated from E14.5 cortical ventricular zone cells. B. A. Reynolds et al., supra; B. A. Reynolds and S. Weiss, supra; WO 9615226; WO 9615224; WO 9609543; WO 9513364; WO 9416718; WO 9410292; WO 9409119. Neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on epidermal growth factor (EGF) for survival. FGF itself is not sufficient for long term growth of neurospheres, though FGF may support their growth transiently. The presently described NEP cells grow in adherent culture, are FGF-dependent, do not express detectable levels of EGF receptors, and are isolated at a stage of embryonic development prior to which it has been possible to isolate neurospheres. Thus, NEP cells appears to represent a multipotent precursor characteristic of the brain stem and spinal cord and peripheral nervous system, while neurospheres may represent a stem cell more characteristic of the cortex.
U.S. Pat. No. 5,589,376, to D. J. Anderson and D. L. Stemple, discloses mammalian neural crest stem cells and methods of isolation and clonal propagation thereof, but fails to disclose cultured NEP cells, cultured lineage restricted precursor cells, and methods of generating, isolating, and culturing thereof. Neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas the present neuroepithelial stem cells differentiate into neurons and glia of the central nervous system (CNS) as well as neural crest stem cells and neurons, glia, and other derivaties of the PNS. This patent also fails to disclose generation of neural crest stem cells from neuroepithelial stem cells.
The present invention is necessary to understand how multipotent neuroepithelial stem cells become restricted to the various neuroepithelial derivatives. In particular, culture conditions that allow the growth and self-renewal of mammalian neuroepithelial stem cells are desirable so that the particulars of the development of these mammalian stem cells can be ascertained. This is desirable because a number of tumors of neuroepithelial derivatives exist in mammals, particularly humans. Knowledge of mammalian neuroepithelial stem cell development is therefore needed to understand these disorders in humans. Additionally, the ability to isolate and grow mammalian neuroepithelial stem cells in vitro allows for the possibility of using such stem cells to treat neurological disorders in mammals, particularly humans. Further, such mammalian neuroepithelial stem cells can be used therapeutically for treatment of certain diseases, e.g. Parkinson""s Disease, such as by transplantation of such cells into an afflicted individual. Moreover, such cells can still further be used for the discovery of genes and drugs that are useful for treating certain diseases. For example, novel genes can be identified by differential display or subtractive hybridization or other screening strategies. Still further, pure NEP stem cell populations according to the present invention can be used to generate and screen antibodies that are specific for these specific cells.
U.S. Ser. No. 08/909,435, filed Jul. 4, 1997, for Isolation of Lineage-Restricted Neuronal Precursors, relates to lineage-restricted precursor cells and methods of making thereof In particular, these cells are neuronal restricted precursors (NRPs) isolated from embryos or NEP stem cells. These neuronal restricted precursors are capable of self-renewal and differentiation into neurons, but not glia (i.e. astrocytes and oligodendrocytes), of the central nervous system. This application is hereby incorporated by reference.
U.S. Ser. No. 08/980,850, filed Nov. 29, 1997, for Lineage Restricted Glial Precursors from the Central Nervous System, relates to glial restricted precursor cells (GRPs) capable of differentiating into oligodendrocytes and two types of astrocytes. These GRPS can also be isolated from embryos or NEP stem cells. This application is also hereby incorporated by reference.
In view of the foregoing, it will be appreciated that isolated populations of mammalian neuroepithelial stem cells and lineage restricted glial precursor cells and methods of generating, isolating, and culturing such cells would be a significant advancement in the art. It will also be appreciated that a method of generating, isolating, and culturing neural crest stem cells and cells of the peripheral nervous system would also be a significant advancement in the art.
It is an object of the present invention to provide isolated (pure) populations of mammalian neuroepithelial stem cells and their progeny in feeder-cell-independent adherent cultures.
It is also an object of the invention to provide populations of mammalian lineage-restricted glial precursor cells and their progeny in feeder-cell-independent adherent cultures.
It is another object of the invention to provide methods of generating, isolating, and culturing mammalian neuroepithelial stem cells and lineage restricted glial precursor cells and their progeny.
It is still another object of the invention to provide methods for the growth and regeneration of neuroepithelial stem cells and lineage restricted glial precursor cells in feeder-cell-independent adherent cultures.
It is yet another object of the invention to provide a method for the generation of lineage restricted glial precursor cells from neuroepithelial stem cells.
It is a still further object of the invention to provide pure differentiated populations of cells derived from neuroepithelial stem cells and lineage restricted glial precursor cells.
It is yet another object of the invention to provide cDNA and cDNA libraries from pure differentiated populations derived from neuroepithelial stem cells and lineage restricted glial precursor cells.
It is still another object of the invention to provide antibodies specific to NEP cells and lineage restricted glial precursor cells.
It is also an object of the invention to provide a method of generating, isolating, and culturing neural crest stem cells and cells of the peripheral nervous system from NEP cells.
These and other objects can be achieved by providing an isolated, pure population of mammalian CNS neuroepithelial stem cells wherein the cells are capable of self-renewal and proliferation in a feeder-cell-independent adherent culture medium and of differentiation to CNS neuronal or glial cells. Preferably, such neuroepithelial stem cells express nestin, but do not express polysialated neural cell adhesion molecule (N-CAM), glial fibrillary acidic protein (GFAP), sulfatide (O4), neurofilament (NF), choline acetyl transferase (ChAT), intermediate filament (xcex2-III tubulin), ganglioside (monoclonal antibody A2B5), or galactocerebroside (Gal-C) immunoreactivities. It is also preferred that such CNS neuronal cells do not express intermediate filament (xcexc-III tubulin) and neurofilament 68 (NF68), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), ganglioside (monoclonal antibody A2B5), sulfatide (O4), or galactocerebroside (Gal-C) immunoreactivies. The neuroepithelial stem cells preferably are further capable of differentiation to glial-restricted precursor cells. Such glial-restricted precursor cells are preferably capable of self-renewal and proliferation in a feeder-cell-independent adherent culture medium and of differentiation to CNS glial cells but not to CNS neuronal cells. These glial-restricted precursor cells preferably express nestin and ganglioside (monoclonal antibody A2B5) immunoreactivies, but do not express glial fibrillary acidic protein (GFAP), sulfatide O4), or galactocerebroside (Gal-C) immunoreactivies.
Another illustrative embodiment of the invention comprises an isolated, pure population of mammalian CNS glial-restricted precursor cells, wherein the glial-restricted precursor cells are capable of self-renewal and proliferation in a feeder-cell-independent adherent culture medium and of differentiation to CNS glial cells but not to CNS neuronal cells.
Still another illustrative embodiment of the invention comprises a method of isolating a pure population of mammalian CNS neuroepithelial stem cells wherein the cells are capable of self-renewal in feeder-cell-independent adherent culture medium and of differentiation to CNS neuronal or glial cells, comprising the steps of:
(a) removing a neural tube from a mammalian embryo at a stage of embryonic development after closure of the neural tube but prior to differentiation of cells in the neural tube;
(b) dissociating cells comprising the neural tube removed from the mammalian embryo;
(c) plating the dissociated cells in feeder-cell-independent culture on a substratum and in a medium configured for supporting adherent growth of the neuroepithelial stem cells, wherein the medium comprises effective amounts of fibroblast growth factor and chick embryo extract; and
(d) incubating the plated cells at a temperature and in an atmosphere conducive to growth of the neuroepithelial stem cells.
Yet another illustrative embodiment of the invention comprises a method of isolating a pure population of mammalian CNS glial-restricted precursor cells wherein the cells are capable of self-renewal in feeder-cell-independent adherent culture medium and of differentiation to CNS glial cells but not CNS neuronal cells, comprising the steps of:
(a) isolating a population of mammalian CNS neuroepithelial stems cells;
(b) incubating the neuroepithelial stem cells in a medium configured for supporting growth of the neuroepithelial stem cells except for lacking an effective amount of chick embryo extract for a period of time sufficient for the cells to begin differentiating;
(c) subjecting the incubated cells to specific antibody capture using an antibody characteristic of glial-restricted precursor cells to result in a captured subpopulation of cells; and
(d) incubating the captured subpopulation of cells in a medium configured for supporting growth thereof comprising effective amounts of fibroblast growth factor and platelet derived growth factor.
Yet another illustrative embodiment of the invention comprises a method of generating a population of mammalian motoneurons comprising the steps of:
(a) isolating a population of mammalian CNS neuroepithelial stems cells;
(b) incubating the neuroepithelial stem cells in a medium that promotes cell proliferation and neuronal differentiation for a period of time sufficient for the cells to begin differentiating; and
(c) isolating motoneurons from said differentiating cells. A preferred medium comprises the use of laminin-coated plates and NEP medium lacking an effective amount of chick embryo extract.
A method for generating mammalian neural crest stem cells comprises inducing neuroepithelial stem cells to differentiate in vitro, thereby generating said neural crest stem cells. Inducing the differentiation of the NEP cells can be carried out by replating the neuroepithelial stem cells on a laminin-coated substrate, withdrawing mitogens such as FGF or chick embryo extract, or adding a dorsalizing agent to the cells. Preferred dorsalizing agents are bone morphogenetic proteins, such as BMP-2, BMP-4, and BMP-7.
A method of generating cells of the peripheral nervous system comprising the steps of:
(a) inducing neuroepithelial stem cells to differentiate in vitro, thereby generating neural crest stem cells;
(b) inducing said neural crest stem cells to differentiate in vitro, thereby generating peripheral nervous system cells.
Neural crest stem cells can be induced to differentiate according to methods well known in the art, such as are described in U.S. Pat. No. 5,589,376, hereby incorporated by reference.